Immunoassay methods using antibodies are used widely due to the high levels of specificity and sensitivity thereof, and the development thereof is expected to increase due to the discovery of monoclonal antibodies in 1975.
Previously radioactive substances, such as (I.sup.125) were used as the means of detection in an immunoassay, but in recent years enzymes and fluorescent substances and the like are used. As a result, expertise in the handling of radioactive substances is no longer required and that is one reason for the accelerated spread of the use of immunoassays.
To achieve a high level of sensitivity in immunoassay, the specific reaction derived from the antigen-antibody reaction must be strong and the other nonspecific reactions must be very weak.
The suppression of the nonspecific reactions is technically important in the perfection of a high sensitivity immunoassay, and therefore, various attempts have been made to achieve same. The most widely practiced means is to include an additive for the suppression of nonspecific reactions into the immunoreaction system. Those means may be classified broadly into two categories: the use of a surfactant, which is nonproteinaceous substance, and the use of a body fluid or a protein solution. As an example of the first method, in Japanese Unexamined Patent Publication (Kokai) No. 57-182169, a soluble polyanion is used as the reaction medium for carrying out the immunoreaction, or in Japanese Unexamined Patent Publication (Kokai) No. 58-187862, a nonspecific adsorption elimination method is disclosed. As examples of the second method, in Japanese Unexamined Patent Publication (Kokai) No. 59-25184 0.1% or more of a hydrophobic protein in the presence of a salt, is used or a reduction of nonspecific adsorption is obtained by the use of mouse ascites, as in Japanese Unexamined Patent Publication (Kokai) No. 61-65162.
The prior art methods as described above have drawbacks and are not fully satisfactory as nonspecific adsorption elimination methods under the present circumstances.
More specifically, the first method frequently will interfere with the specific reaction resulting in an assay system having low sensitivity. Although the use of a hydrophobic protein has been mentioned as a second method, according to investigations made by the instant inventors, hydrophobicity had substantially no nonspecific adsorption elimination effect.
Further, when mouse ascites are used as in the second method, a the problem arises in the reproducibility of the components of the ascites, and the specific reaction may be reduced by these components, contrary to the object of immunoassay per se.
Nevertheless, there are great expectations for the achievement of high sensitivity by an immunoreaction on a membrane, such as nitrocellulose or the like, through a concentration of the antigen on the membrane rather than in the above-mentioned solution; and this technique has become widely used. report in J. Biochem. Biophys. Methods 12, 271 to 279 (1986), the observation of a striking reduction in the nonspecific reactions by blocking a membrane using a 10% suspension of skim milk.
As the solid-phase reaction blocking agent, Ahmad et al. used skim milk. In the J. Clin. Microbiol. 23, 3, 563 to 567 (1986), Ahmad et al. measured the anti-pseudorabies virus antibodies in the serum of patients by blocking a nitrocellulose membrane carrying immobilized pseudorabies virus using a 5% skim milk solution.
However, if a 5% to 10% skim milk suspension is used as the blocking agent, as stated in the report by Baldo et al, there is a large decline in the specific reaction and thus a loss of one of the two essential conditions for high sensitivity of an immunoassay (a high specific reaction derived from the antigen-antibody reaction) and a resulting inability to achieve a highly sensitive assay.
The reason for the inhibition of the specific reaction is that skim milk is insoluble in water at that concentration and essentially is a suspension. When observed under a microscope, large insoluble particles of the skim milk cover the antigen preventing the antibodies from approaching the same, and as a result, considerably inhibiting the antigen-antibody reaction.
The use of skim milk as a blocking agent in the prior art is used commonly in the above-mentioned solid phase immunoreaction but not often in the case of an immunoreaction in the solution phase.
In the past, there has only been one report on the use of skim milk as an additive in an immunoreaction in the solution phase; that report was made by Kuroki et al. in Pedialr. Res. 19, 1017 (1985), a 2% skim milk suspension was used for an assay of lung-surfactant apoprotein in amniotic fluid. Two major problems arose in the use of skim milk in this method; first, that the suspension of skim milk was used so the specific immunoreaction was suppressed for the reason mentioned above, and second, that in the 2% skim milk suspension, precipitation occurs after two or three weeks storage in a refrigerator and it is impossible to redissolve the precipitate, thus resulting in a loss of the ability for inhibiting nonspecific reactions. In other words, the suspension must be used immediately after preparation or the function for inhibiting nonspecific reactions is lost and thus it is most unsatisfactory as a reagent.